Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

• the isolated polynucleotide disclosed above encodes a polypeptide that has activity as measured by cell proliferation, activation of transcription of a reporter gene, or wherein the polypeptide encoded by the polynucleotide further binds to an antibody, wherein the antibody is raised to a polypeptide comprising a sequence of amino acids from the group consisting of: (a) the polypeptide comprising amino acid number 20 (Ala) to 227 (Pro) of SEQ ID NO:2; (b) the polypeptide comprising amino acid number 20 (Ala) to 519 (Glu) of SEQ ID NO:2; (c) the polypeptide comprising amino acid number 20 (Ala) to 543 (Leu) of SEQ ID NO:2; (d) the polypeptide comprising amino acid number 544 (Lys) to 732 (Val) of SEQ ID NO:2; (e) the polypeptide comprising amino acid number 544 (Lys) to 649 (He) of SEQ ID NO:46; (a) the
• affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
• Affinity tags include a poly- histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl.
• operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
• ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
• a "soluble receptor” is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis. Soluble receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
• any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
• the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
• WSN can, in some circumstances, encode arginine
• MGN degenerate codon for arginine
• AGY serine
• preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ HD NO:4, SEQ HD NO:47 and SEQ DD NO:55 serve as templates for optimizing expression of zcytorl7 polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
• the isolated polynucleotides will hybridize to similar sized regions of SEQ DD NO:l, SEQ DD NO:45, or SEQ DD NO:54; or SEQ DD NO:57 and SEQ DD NO:93; or a sequence complementary thereto, under stringent conditions.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextian sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
• such stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC.
• An alternative way to prepare a full-length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3' and 5' short overlapping complementary regions (6 to 10 nucleotides) are annealed, large gaps still remain, but the short base-paired regions are both long enough and stable enough to hold the structure together. The gaps are filled and the DNA duplex is completed via enzymatic DNA synthesis by E. coli DNA polymerase I. After the enzymatic synthesis is completed, the nicks are sealed with T4 DNA ligase. Double-stranded constructs are sequentially linked to one another to form the entire gene sequence which is verified by DNA sequence analysis.
• the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zcytorl7 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zcytorl7 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
• Cytokine receptor subunits are characterized by a multi-domain structure comprising an extracellular domain, a transmembrane domain that anchors the polypeptide in the cell membrane, and an intracellular domain.
• the extracellular domain may be a ligand-binding domain
• the intracellular domain may be an effector domain involved in signal transduction, although ligand-binding and effector functions may reside on separate subunits of a multimeric receptor.
• the ligand-binding domain may itself be a multi-domain structure.
• zcytorl7 sequence suggests that it is a member of the same receptor subfamily as the gpl30, LfF, IL-12, WSX-1, IL-2 receptor ⁇ -subunit, IL- 3, IL-4, and IL-6 receptors.
• Certain receptors in this subfamily e.g., G-CSF
• Other members of the subfamily e.g., gpl30, IL-6, 1L-11, and LIF receptors
• combine with a second subunit Termed a ⁇ - subunit to bind ligand and transduce a signal.
• Specific ⁇ -subunits associate with a plurality of specific cytokine receptor subunits.
• sequences disclosed in SEQ DD NO:l, SEQ DD NO:45 and SEQ DD NO:53 represent alleles of human zcytorl7 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
• the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zcytorl7.
• the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzvmol. 183:63 (1990).
• Variant zcytorl7 polypeptides or substantially homologous zcytorl7 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
• variant zcytorl7 soluble receptors such as those shown in SEQ DD NO: 18 and SEQ DD NO:22 can be isolated.
• polypeptides may include additional amino acids from, for example, part or all of the transmembrane and intracellular domains.
• polypeptides may also include additional polypeptide segments as generally disclosed herein such as labels, affinity tags, and the like.
• Cultured mammalian cells are suitable hosts within the present invention.
• Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
• Drag selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
• a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drag, such as G-418 or the like.
• Suitable media are Sf900 DTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zcytorl7 polypeptide from the supernatant can be achieved using methods described herein.
• Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
• DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
• the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUGl or AUG2).
• DHAS dihydroxyacetone synthase
• FMD formate dehydrogenase
• CAT catalase
• a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AER.C; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine.
• Cells expressing functional zcytorl7 are used within screening assays.
• a variety of suitable assays are known in the art. These assays are based on the detection of a biological response in the target cell.
• One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the reduction or metabolic breakdown of Alymar BlueTM (AccuMed, Chicago, EL) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth.
• An alternative assay format uses cells that are further engineered to express a reporter gene.
• the reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene.
• a preferred promoter element in this regard is a serum response element, STAT or SRE (see, for example, Shaw et al., Cell 56:563-572, 1989).
• a preferred such reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem.
• Media samples from the transfected cells are then assayed, with subsequent division of pools, retransfection, subculturing, and re-assay of positive cells to isolate a clonal cell line expressing the ligand.
• Media samples conditioned by kidney, liver, spleen, thymus, other lymphoid tissues, or T-cells are preferred sources of ligand for use in screening procedures.
• the intracellular domain of zcytorl7 comprising approximately residues 544 (Lys) to 732 (Val) of SEQ DD NO:2, residues 544 (Lys) to 649 (He) of SEQ HD NO:46, or residues 557 (Lys) to 662 (He) of SEQ HD NO:54, or residues 551 (Lys) to 662 (Cys) of SEQ HD NO:57 is joined to the ligand-binding domain of a second receptor. It is preferred that the second receptor be a hematopoietic cytokine receptor, such as mpl receptor (Souyri et al., Cell 63:1137-1147, 1990).
• a second class of hybrid receptor polypeptides comprise the extracellular (ligand-binding) domain (approximately residues 20 (Ala) to 519 (Glu) of SEQ DD NO:2 and SEQ DD NO:46; approximately residues 33 (Ala) to 532 (Glu) of SEQ DD NO:54) or cytokine-binding domain of zcytorl7 (approximately residues 20 (Ala) to 227 (Pro) of SEQ DD NO:2 and SEQ DD NO:46; or approximately residues 33 (Ala) to 240 (Pro) of SEQ DD NO:54; approximately residues 46 (Val) to 533 (Glu) of SEQ DD NO:57; or approximately residues 46 (Val) to 533 (Trp) of SEQ DD NO:93) with a cytoplasmic domain of a second receptor, preferably a cytokine receptor, and a transmembrane domain.
• a second receptor preferably a cytokine receptor,
• the extracellular domain be prepared in a form substantially free of transmembrane and intracellular polypeptide segments.
• ligand-binding polypeptide fragments within the zcytorl7 cytokine binding domain can also serve as zcytorl7 soluble receptors for uses described herein.
• the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide or a zcytorl7 secretory peptide.
• the molecules of the present invention have particular use in the monocyte/macrophage arm of the immune system.
• interferon gamma IFN ⁇
• IFN ⁇ interferon gamma
• THP-1 cells ATCC No. TD3-202
• zcytorl7 upon activation of THP-1 cells
• TD3-202 THP-1 cells
• Macrophages play a central role in the immune response by presenting antigen to lymphocytes and play a supportive role as accessory cells to lymphocytes by secreting numerous cytokines.
• the monocyte-macrophage system is prominently involved in several lipid-storage diseases (sphingolipidoses) such as Gaucher's disease. Resistance to infection can be impaired because of a defect in macrophage function, which could be treated by agonists to zcytorl7 such as anti- zcytorl7 antibodies and binding partners, as well as the natural ligand.
• sphingolipidoses lipid-storage diseases
• Resistance to infection can be impaired because of a defect in macrophage function, which could be treated by agonists to zcytorl7 such as anti- zcytorl7 antibodies and binding partners, as well as the natural ligand.
• anti-zcytorl7 antibodies and binding partners can be conjugated to molecules such as toxic moieties and cytokines, as described herein to direct the killing of leukemia monocytic cells.
• molecules such as toxic moieties and cytokines, as described herein to direct the killing of leukemia monocytic cells.
• Zcytorl7 has been shown to be upregulated in monocyte cells, and may be involved in regulating inflammation.
• polypeptides of the present invention can be assayed and used for their ability to modify inflammation, or can be used as a marker for inflammation.
• Methods to determine proinflammatory and antiinflammatory qualities of zcytorl7 are known in the art and discussed herein.
• it may be involved in up-regulating the production of acute phase reactants, such as serum amyloid A (SAA), ⁇ l-antichymotrypsin, and haptoglobin, and that expression of zcytorl7 ligand may be increased upon injection of lipopolysaccharide (LPS) in vivo that are involved in inflammatory response (Dumoutier, L.
• SAA serum amyloid A
• ⁇ l-antichymotrypsin ⁇ l-antichymotrypsin
• haptoglobin haptoglobin
• particular embodiments of the present invention are directed toward use of soluble zcytorl7 heterodimers as antagonists in inflammatory and immune diseases or conditions such as pancreatitis, type I diabetes (DDDM), pancreatic cancer, pancreatitis, Graves Disease, inflammatory bowel disease (D3D), Crohn's Disease, colon and intestinal cancer, diverticulosis, autoimmune disease, sepsis, organ or bone marrow transplant; inflammation due to trauma, powery or infection; amyloidosis; splenomegaly; graft versus host disease; and where inhibition of inflammation, immune suppression, reduction of proliferation of hematopoietic, immune, inflammatory or lymphoid cells, macrophages, T-cells (including Thl and Th2 cells, CD4+ and CD8+ cells), suppression of immune response to a pathogen or antigen.
• DDDM type I diabetes
• pancreatic cancer pancreatitis
• Graves Disease inflammatory bowel disease
• Crohn's Disease colon and
• T-cell specific leukemias, lymphomas, and carcinoma may be treated with monoclonal antibodies to zcytorl7-comprising soluble receptors of the present invention.
• Soluble zcytorl7 monomeric, homodimeric, heterodimeric and multimeric polypeptides described herein can be used to neutralize/block zcytorl7 ligand activity in the treatment of autoimmune disease, atopic disease, NDDDM, pancreatitis and kidney dysfunction as described above.
• a soluble form of zcytorl7 may be used to promote an antibody response mediated by T cells and/or to promote the production of IL-4 or other cytokines by lymphocytes or other immune cells.
• tumor cells passaged in culture are implanted into mice of the same strain as the tumor donor.
• the cells will develop into tumors having similar characteristics in the recipient mice, and metastasis will also occur in some of the models.
• Appropriate tumor models for our studies include the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No. CRL-6323), amongst others.
• adenoviras can accommodate relatively large DNA inserts; (ii) can be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with a large number of different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenovirases are stable in the bloodstream, they can be administered by intravenous injection.
• adenoviras vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
• the essential El gene has been deleted from the viral vector, and the viras will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
• the host cell the human 293 cell line is exemplary.
• adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the viras cannot replicate in the host cells.
• the adenovirus system can also be used for protein production in vitro.
• the cells By culturing adenoviras-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
• adenoviras vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Gamier et al., Cytotechnol. 15:145-55, 1994).
• Reporter gene constructs of this type are known in the art, and will generally comprise a zcytorl7-DNA response element operably linked to a gene encoding an assay detectable protein, such as luciferase.
• DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem.
• a method of identifying agonists of zcytorl7 polypeptide comprising providing cells responsive to a zcytorl7 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a increase in a cellular response of the second portion of the cells as compared to the first portion of the cells.
• third cell containing the reporter gene construct described above, but not expressing zcytorl7 receptor, can be used as a control cell to assess non-specific, or non-zcytorl7-mediated, stimulation of the reporter.
• Agonists, including the natural ligand, are therefore useful to stimulate or increase zcytorl7 polypeptide function.
• An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument may be advantageously employed (e.g., BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ; or SELDF M technology, Ciphergen, Inc., Palo Alto, CA).
• a ligand-binding receptor or an antibody, one member of a complement/ anti-complement pair
• a commercially available biosensor instrument may be advantageously employed (e.g., BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ; or SELDF M technology, Ciphergen, Inc., Palo Alto, CA).
• BIAcoreTM Pharmacia Biosensor, Piscataway, NJ
• SELDF M technology Ciphergen, Inc., Palo Alto, CA
• Such receptor, antibody, member of a complement/anti- complement pair or fragment is immobilized onto the surface of
• a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextian fibers that are attached to gold film within the flow ceH.
• a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
• This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
• Polypeptides comprising a larger portion of a zcytorl7 polypeptide, i.e., from 30 to 100 residues up to the entire length of the amino acid sequence are included.
• Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein.
• humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced. Moreover, human antibodies can be produced in transgenic, non-human animals that have been engineered to contain human immunoglobulin genes as disclosed in WIPO Publication WO 98/24893. It is preferred that the endogenous immunoglobulin genes in these animals be inactivated or eliminated, such as by homologous recombination.
• random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
• a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
• Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al, US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
• binding peptides which interact with zcytorl7 polypeptides can be used for tagging cells that express the receptor; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drags, toxins, radionuclides and the like. These binding peptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding peptides can also be used for diagnostic assays for determining circulating levels of zcytorl7 polypeptides; for detecting or quantitating soluble zcytorl7 polypeptides as marker of underlying pathology or disease.
• binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
• anti-zcytorl7 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zcytorl7 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
• known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family (e.g., gpl30, LIF, WSX-1 and IL12 receptors). Screening can also be done using non-human zcytorl7, and zcytorl7 mutant polypeptides.
• antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zcytorl7 polypeptides.
• antibodies raised to zcytorl7 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zcytorl7 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides ( " Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995).
• assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zcytorl7 protein or polypeptide.
• Antibodies to zcytorl7 may be used for tagging cells that express zcytorl7, such as cells that naturally express zcytorl7 such as monocyte and prostate cells, as well as cells that are transformed with zcytorl7; for isolating zcytorl7 by affinity purification; for diagnostic assays for determining circulating levels of zcytorl7 polypeptides; for detecting or quantitating soluble zcytorl7 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zcytorl7 activity in vitro and in vivo.
• Antibodies to zcytorl7 are useful for tagging cells that express the receptor and assaying Zcytorl7 expression levels, for affinity purification, within diagnostic assays for determining circulating levels of soluble receptor polypeptides, analytical methods employing fluorescence-activated cell sorting. Divalent antibodies may be used as agonists to mimic the effect of a zcytorl7 ligand.
• Antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• antibodies or binding polypeptides which recognize zcytorl7 of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (i.e., a zcytorl7 receptor).
• anti-zcytorl7 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the zcytorl7 molecule.
• Suitable detectable molecules may be directly or indirectly attached to polypeptides that bind zcytorl7 ("binding polypeptides," including binding peptides disclosed above), antibodies, or bioactive fragments or portions thereof.
• Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
• the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the binding polypeptide or antibody portion.
• biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
• zcytorl7 binding polypeptide-cytokine or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood, lymphoid, colon, and bone marrow cancers), if the binding polypeptide-cytokine or anti-zcytorl7 antibody targets the hyperproliferative cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentiation of cytokine.
• Suitable anti-zcytorl7 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells.
• Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.
• GM-CSF granulocyte-macrophage colony-stimulating factor
• zcytorl7 binding polypeptide or antibody fusion proteins described herein can be used for enhancing in vivo killing of target tissues by directly stimulating a zcytorl7-modulated apoptotic pathway, resulting in cell death of hyperproliferative cells expressing zcytorl7.
• bioactive binding polypeptide or antibody conjugates described herein can be delivered orally, intravenously, intraarterially or intraductally, or may be intioduced locally at the intended site of action.
• Four-helix bundle cytokines that bind to cytokine receptors as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body.
• Therapeutic utility includes treatment of diseases which require immune regulation including autoimmune diseases, such as, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes.
• Zcytorl7 antagonists or agonists may be a potential therapeutic in suppressing the immune system which would be important for reducing graft rejection or in prevention of graft vs. host disease.
• zcytorl7 antagonists or agonists including soluble receptors, anti- zcytorl7 receptor antibodies and the natural ligand may activate the immune system which would be important in boosting immunity to infectious diseases, treating immunocompromised patients, such as HlV-r patient, or in improving vaccines.
• Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex viras (HSV), papillomavirus, Epstein Barr virus (EBV), adenoviras, adeno-associated viras (AAV), and the like.
• HSV herpes simplex viras
• EBV Epstein Barr virus
• AAV adeno-associated viras
• Defective viruses which entirely or almost entirely lack viral genes, are preferred.
• a defective viras is not infective after introduction into a cell.
• Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
• Examples of particular vectors include, but are not limited to, a defective herpes simplex viras 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
• Antisense methodology can be used to inhibit zcytorl7 gene transcription, such as to inhibit cell proliferation in vivo.
• Polynucleotides that are complementary to a segment of a zcytorl7-encoding polynucleotide e.g., a polynucleotide as set froth in SEQ HD NO:l, SEQ HD NO:45, SEQ HD NO:53, or SEQ DD NO:57
• Such antisense polynucleotides are used to inhibit expression of zcytorl7 polypeptide-encoding genes in cell culture or in a subject.
• the present invention also provides reagents that will find use in diagnostic applications.
• the zcytorl7 gene a probe comprising zcytorl7 DNA or RNA or a subsequence thereof can be used to determine if the zcytorl7 gene is present on chromosome 5 or if a mutation has occurred.
• Zcytorl7 is located at the 5ql 1 region of chromosome 5 (See, Example 4).
• Detectable chromosomal aberrations at the zcytorl7 gene locus include, but are not limited to, aneuploidy, gene copy number changes, loss of heterogeneity (LOH), translocations, insertions, deletions, restriction site changes and rearrangements.
• Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
• RFLP restriction fragment length polymorphism
• STR short tandem repeat
• Exons are readily determined by one of skill in the art by comparing zcytorl7 sequences (e.g., SEQ HD NO:54) with the human genomic DNA for zcytorl7 (Genbank Accession No. AQ002781).
• zcytorl7 sequences e.g., SEQ HD NO:54
• human genomic DNA for zcytorl7 Genbank Accession No. AQ002781.
• the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient are known in the art.
• Mutations associated with the zcytor 17 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
• standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-as
• the mouse models of the present invention are particularly relevant as, immune system models, inflammation or tumor models for the study of cancer biology and progression. Such models are useful in the development and efficacy of therapeutic molecules used in human immune diseases, inflammation and cancers. Because increases in zcytorl7 expression, as well as decreases in zcytorl7 expression are associated with monocytes, monocyte activation, and prostate cells, and may be associated with inflammation and cancers, both transgenic mice and knockout mice would serve as useful animal models for human disease. Moreover, in a preferred embodiment, zcytorl7 transgenic mouse can serve as an animal model for specific diseases, particularly those associated with monocytes. Moreover, transgenic mice expression of zcytorl7 antisense polynucleotides or ribozymes directed against zcytorl7, described herein, can be used analogously to transgenic mice described above.
• Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of zcytorl7 can be used as standards or as "unknowns" for testing purposes.
• zcytorl7 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constracts for bacterial, viral, and/or mammalian expression, including fusion constracts, wherein zcytorl7 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zcytorl7 polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
• Zcytor 17 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional stracture in atomic detail, nuclear magnetic resonance spectroscopy to reveal the stracture of proteins in solution.
• analytical skills such as mass spectrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional stracture in atomic detail, nuclear magnetic resonance spectroscopy to reveal the stracture of proteins in solution.
• a kit containing the zcytorl7 can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the specific protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of zcytorl7 would
• the zcytorl7 gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities and other educational entities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum. Such educational kits containing the zcytorl7 gene, polypeptide or antibody are considered within the scope of the present invention.
• Primers for 3' RACE were designed using the 5' RACE product (SEQ DD NO:9) obtained above.
• a 3' RACE product was generated using a human prostate in- house cDNA library as a template and oligonucleotides ZC28,481 (SEQ DD NO: 10) and
• ZC6,346 (SEQ DD NO: 11) as primers. This first-round 3' RACE PCR reaction was ran under the conditions described in Example 1A. An aliquot of 3' RACE PCR product was removed and analyzed on a 1.0% agarose gel. Multiple bands were seen on the gel.
• the remaining 3' RACE PCR product was ethanol precipitated and diluted 1:40.
• a second-round nested 3' RACE PCR reaction was run to amplify template cDNA sequence.
• This PCR reaction used oligonucleotides ZC28,480 (SEQ DD NO: 12) and ZC26,405 (SEQ DD NO: 13), which were designed to anneal to sequence internal of ZC28,481 (SEQ DD NO: 10) and ZC6,346 (SEQ DD NO:l 1).
• This nested PCR reaction was ran as disclosed above.
• the resulting DNA products were electtophoresed on a 1.0% agarose gel and a prominent band at approximately 2100 bp was seen.
• a third- round nested 3' RACE PCR reaction was run to amplify template cDNA sequence.
• This PCR reaction used oligonucleotides ZC27,895 (SEQ DD NO: 14) and ZC5,020 (SEQ DD NO: 15), which were designed to anneal to sequence internal of ZC28,480 (SEQ DD NO: 12) and ZC26,405 (SEQ DD NO: 13).
• This nested PCR reaction was run as disclosed above. The resulting PCR products were electrophoresed on a 1.0% agarose gel and a prominent band at approximately 2000 bp was seen. The DNA band was gel purified and sequenced.
• a second 5' RACE for zcytorl7 identified an alternative full-length zcytor!7
• a 5' RACE product was generated using a WI-38 cDNA library as a template and oligonucleotides ZC12/701 (SEQ DD NO:5)and ZC27,899 (SEQ DD NO:8) as primers.
• WI-28 is an in-house cDNA library generated from a human embryonic lung cell line (ATCC No. CRL-75). The PCR reaction used approximately 1 ⁇ g of of plasmid
• probes A-D The four probes, herein designated probes A-D, were amplified using the following primer pairs: (A) ZC28,575 (SEQ DD NO:23), ZC27,899 (SEQ DD NO:24); (B) ZC27,895 (SEQ DD NO:64), ZC28,917 (SEQ DD NO:73); (C) ZC28,916 (SEQ DD NO:75), ZC28,918 (SEQ DD NO:76); and (D) ZC28,916 (SEQ DD NO:75), ZC29,122 (SEQ DD NO:65).
• Example 11 Constraction of Mammalian Expression Vectors That Express zcytor!7 Soluble Receptors: zcytorl7CEE, zcytorl7CFLG, zcvtorl7CHIS and zcytor!7-Fc4 A. Constraction of zcytor!7 Mammalian Expression Vector containing zcytorl7CEE, zcvtorl7CFLG and zcvtor!7CHIS
• a human HPVS in-house cDNA library was used as a template and PCR amplification was performed as follows: 30 cycles at 94°C for 1 minute, 65°C for 1 minute, 72°C for 1.5 minutes, then 72°C for 7 minutes; 10°C soak.
• the PCR reaction was ethanol precipitated and digested with EcoRI and BamHI restriction enzymes.
• the digested PCR product was gel purified on a 1.0% agarose gel and the approximately 1500 bp band excised. This band was then re-amplified using identical primers with the following cycling: 30 cycles at 94°C for 1 minute, 65°C for 1 minute, 72°C for 3 minutes, then 72°C for 7 minutes; 10°C soak.
• a 1566 base pair fragment was generated by PCR, containing the extracellular domain of human zcytor 17 and the first two amino acids of Fc4 (Glu and Pro) with Fsel and BglE sites coded on the 5' and 3' ends, respectively.
• This PCR fragment was generated using primers ZC29,157 (SEQ DD NO:70) and ZC29,150 (SEQ DD NO:71) by amplification from a plasmid containing the extracellular domain of human zcytorl7 (pZp9zcytorl7CEE) (Example 11 A).
• Two ⁇ l of the ligation mix was electroporated in 37 ⁇ l DH10B electrocompetent E. coli (Gibco) according to the manufacturer's directions.
• the transformed cells were diluted in 400 ⁇ l of LB media and plated onto LB plates containing 100 ⁇ g/ml ampicillin. Clones were analyzed by restriction digests and positive clones were sent for DNA sequencing to confirm the sequence of the fusion construct.
• 1 ⁇ l of a positive clone was transformed into 37 ⁇ l of DH10B electrocompetent E. coli and streaked on a LB/amp plate.
• pEZE-2hzcytorl7Fc4 plasmid DNA Twenty ⁇ g of pEZE-2hzcytorl7Fc4 plasmid DNA (Example IIB) was linearized by restriction digestion with Fspl, a restriction enzyme that cuts once within the pEZE-2 vector and does not disturb genes necessary for expression. 200 ⁇ g of sheared salmon sperm DNA was added as carrier DNA and then the DNA was precipitated by addition of 0.1 volumes of 3M Sodium Acetate pH 5.2 and 2.2 volumes ethanol followed by a 15 minute ice incubation and microcentrifugation at 4°C.
• the eluted protein was collected into 0.1 ml of 2 M Tris, pH 8.5.
• An expression plasmid containing a polynucleotide encoding a zcytor 17 soluble receptor fused C-terminally to maltose binding protein (MBP) was constructed via homologous recombination.
• the fusion polypeptide contains an N-terminal approximately 388 amino acid MBP portion fused to any of the zcytorl7 soluble receptors described herein.
• a fragment of zcytorl7 cDNA (SEQ HD NO:l, SEQ HD NO:45, SEQ HD NO: 17 or SEQ ED NO:21) was isolated using PCR as described herein.
• Two primers were used in the production of the zcytorl7 fragment in a standard PCR reaction: (1) one containing about 40 bp of the vector flanking sequence and about 25 bp corresponding to the amino terminus of the zcytor 17, and (2) another containing about 40 bp of the 3' end corresponding to the flanking vector sequence and about 25 bp corresponding to the carboxyl terminus of the zcytorl7.
• Two ⁇ l of the 100 ⁇ l PCR reaction was ran on a 1.0% agarose gel with 1 x TBE buffer for analysis, and the expected approximately fragment was seen.
• the remaining PCR reaction was combined with the second PCR tube and precipitated with 400 ⁇ l of absolute ethanol.
• the precipitated DNA used for recombining into the Smal cut recipient vector pTAP170 to produce the construct encoding the MBP-zcytorl7 fusion, as described below.
• Competent yeast cells (S. cerevisiae) were combined with about 10 ⁇ l of a mixture containing approximately 1 ⁇ g of the zcytor 17 receptor PCR product above, and 100 ng of Smal digested pTAP98 vector, and electroporated using standard methods and plated onto URA-D plates and incubated at 30°C.
• Ura+ yeast transformants from a single plate were picked, DNA was isolated, and transformed into electrocompetent E. coli cells (e.g., MC1061, Casadaban et. al. X Mol. Biol. 138, 179-207), and plated on MM/CA +AMP 100 mg/L plates (Pryor and Leiting, Protein Expression and Purification 10:309- 319, 1997).using standard procedures. Cells were grown in MM/CA with 100 ⁇ g/ml Ampicillin for two hours, shaking, at 37°C. 1ml of the culture was induced with ImM IPTG.
• the frozen cells were thawed and diluted 1:10 in a protease inhibitor solution (Extraction buffer) prior to lysing the cells and releasing the huzcytorl7/MBP- 6H soluble receptor protein.
• the diluted cells contained final concentrations of 20 mM Tris (JT Baker, Philipsburg, NJ) 100 mM Sodium Chloride (NaCl, Mallinkrodt, Paris, KY), 0.5 mM pheynlmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., St. Louis, MO), 2 ⁇ g/ml Leupeptin (Fluka, Switzerland), and 2 ⁇ g/ml Aprotinin (Sigma).
• Example 15 Zcytorl7 receptor Monoclonal Antibodies Zcytor 17 soluble receptor Monoclonal Antibodies Zcytor 17 soluble receptor Monoclonal antibodies are prepared by immunizing female BalbC mice with the purified recombinant soluble zcytorl7 proteins described herein. The mice are each given an initial intraperitoneal (IP) injection of 20 ug of purified protein in Complete Freund's Adjuvant (Pierce, Rockford, IL) followed by booster IP injections of 10 ug purified protein in Incomplete Freund's Adjuvant every two weeks. Seven to ten days after the administration of the third booster injection, the animals are bled and the serum is collected, and antibody titer assessed.
• IP intraperitoneal
• Preferred ligands are those that can bind class 1 heterodimeric cytokine receptors such as, gpl30, LIF, IL-12, IL-2, IL-4, IL-7, E.-9, 1L-15, zalphall Ligand (IL- 21) (commonly owned US Pat. Application No. 09/522,217), TSLP (Levine, SD et al., ibid.; Isaksen, DE et al., ibid.; Ray, RJ et al., ibid.; Friend, SL et al., ibid.).
• class 1 heterodimeric cytokine receptors such as, gpl30, LIF, IL-12, IL-2, IL-4, IL-7, E.-9, 1L-15, zalphall Ligand (IL- 21) (commonly owned US Pat. Application No. 09/522,217), TSLP (Levine, SD et al., i
• the resulting vector is sequenced to confirm that the zcytorl7/IgG gamma 1 fusion (zcytor 17/Chl IgG) is correct.
• a separate constract having a heterodimeric cytokine receptor subunit extracellular domain fused to K light is also constracted as above.
• the cytokine receptor/human K light chain construction is performed as above by PCRing from, e.g., a lymphocyte cDNA library (Clontech) using standard methods, and oligos that provide EcoRI and Kpnl restriction sites.
• the resulting PCR product is digested with EcoRI and Kpnl and then ligating this product into a previously EcoRI and Kpnl digested and band- purified Zem228R/human K light chain vector described above.
• the resulting vector is sequenced to confirm that the cytokine receptor subunit/human K light chain fusion is correct.
• Approximately 15 ⁇ g of each of vectors above, are co-transfected into mammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) using LipofectaminePlusTM reagent (Gibco/BRL), as per manufacturer's instructions.
• the transfected cells are selected for 10 days in DMEM + 5%FBS (Gibco/BRL) containing 1 ⁇ M of methotrexate (MTX) (Sigma, St. Louis, MO) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days.
• the resulting pool of transfectants is selected again in 10 ⁇ m of MTX and 0.5 mg/ml G418 for 10 days.
• MPL-class I cytokine receptor fusion that signals in the presence of the TPO ligand, determines which heterodimeric cytokine receptor subunits are required for zcytorl7 receptor signaling.
• Use of MPL-receptor fusions for this purpose alleviates the requirement for the presence of a natural ligand for the zcytorl7 receptor.
• MPL-class I cytokine receptor fusions are made as per Example 5 using the extracellular domain and transmembrane domains of the MPL receptor and the intracellular signaling domain of the desired class I cytokine receptor.
• Receptor complexes include but are not limited to zcytorl7 receptor in combination with an MPL-cytokine receptor fusion comprising a gpl30, LIF, IL-12, or WSX-1 component, or one or more of the IL-2 receptor components (IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ ), zcytor 17 receptor with one or more of the IL-4/TL-13 receptor family receptor components (IL-4R ⁇ , IL-13R ⁇ , E.-13R ⁇ '), as well as other Interleukin receptors (e.g., IL-15 R ⁇ , E 7R ⁇ , E--9R ⁇ , IL-21R (Zalphall receptor)).
• MPL-cytokine receptor fusion comprising a gpl30, LIF, IL-12, or WSX-1 component
• IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ zcytor 17 receptor with one or more of the IL-4/TL-13 receptor family receptor components (IL-4R ⁇ , IL
• a BaF3/MPL-class I cytokine receptor cell line can be constracted to control for MPL-class I cytokine receptor homodimerization effects for those class I cytokine receptors known to signal upon homodimerization.
• the TPO in the presence of the correct receptor complex is expected to increase proliferation of the BaF3/MPL-zcytorl7/MPL-class I cytokine receptor cell line approximately '5 fold over background or greater in the presence of TPO.
• An exemplary luciferase reporter mammalian expression vector is the KZ134 plasmid which is constracted with complementary oligonucleotides ZC 12,749 (SEQ HD NO:43) and ZC12,748 (SEQ ED NO:44) that contain STAT transcription factor binding elements from 4 genes.
• a modified c-fos Sis inducible element m67SIE, or hSIE
• m67SIE or hSIE
• the KZ134 plasmid is used to stably transfect BHK, or BaF3 cells, using standard transfection and selection methods, to make a BHK/KZ134 or BaF3/KZ134 cell line respectively.
• the bioassay cell line is tiansfected with zcytorl7 receptor alone, or co- transfected with zcytor 17 receptor along with one of a variety of other known receptor subunits.
• Receptor complexes include but are not limited to zcytor 17 receptor only, various combinations of zcytorl7 receptor with gpl30, LIF, IL-12, or WSX-1 receptor subunits, or one or more of the IL-2 receptor components (IL-2R ⁇ , E_,-2R ⁇ , E,-2R ⁇ ), zcytorl7 receptor with one or more of the JL-4/ L-13 receptor family receptor components (TL-4R ⁇ , IL-13R ⁇ , IL ⁇ 13R ⁇ '), as well as other Interleukin receptors (e.g., IL-15 R ⁇ , E_,-7R ⁇ , H_,-9R ⁇ , IL-21R (zalphall)).
• IL-15 R ⁇ , E_,-7R ⁇ , H_,-9R ⁇ , IL-21R (zalphall) Interleukin receptors
• Each independent receptor complex cell line is then assayed in the presence of cytokine-conditioned media or purified cytokines and luciferase activity measured using routine methods.
• the untransfected bioassay cell line serves as a control for the background luciferase activity, and is thus used as a baseline to compare signaling by the various receptor complex combinations.
• the conditioned medium or cytokine that binds the zcytorl7 receptor in the presence of the correct receptor complex is expected to give a luciferase readout of approximately 5 fold over background or greater.
• BaF3 cells for electroporation were washed twice in PBS, pH 7.2 (Gibco- BRL) and then resuspended in PBS and a cell density of 10 7 cells/ml.
• One ml of resuspended BaF3 cells was mixed with 30 ⁇ g of the pZP7PX/zcytorl7 plasmid DNA and transferred to separate disposable electroporation chambers (Gibco-BRL).
• the cells were then given 2 serial shocks (800 lFad/300V.; 1180 lFad/300V.) delivered by an electroporation apparatus (CELL-PORATORTM; Gibco-BRL).
• the electroporated cells were then transferred to 20 mis of complete media and placed in an incubator for 48 hours (37 °C, 5% CO 2 ). The cells were then spun down and resuspended in 20 mis of complete media containing 2 ⁇ g/ml Puromycin (ClonTech) Selection in a T75 flask to isolate the puromycin resistant pool. Clonal lines of the transfected BaF3 cells, hereinafter called BaF3/zcytorl7 cells, were isolated as described below.
• BaF3/zcytorl7 cells were counted in a hemocytometer, and plated at 1 cell/well, 0.5 cell/well, 0.1 cell/well, and 0.01 cell/well, in a volume of 100 ⁇ l/well in complete media containing 2 ⁇ g/ml Puromycin. 15 clones were scaled up to T75 flasks, and 5 clones were assayed for RNA production. Cells were washed once with PBS and counted with a hemocytometer. 5 x 10 6 cells were spun down and the media removed. Untransfected BaF3 cells were also counted and pelleted. Four of the pellets were frozen at -80°C overnight.
• the filters were pre-washed at 65°C in pre-wash buffer consisting of 0.25X SSC, 0.25% SDS and ImM EDTA. Cell debris was manually removed using Kimwipes® (Kimberly-Clark) and the solution was changed 3 times over a period of 1 hour. Filters were air dried and stored at room temperature until needed. The filters were then prehybridized for approximately 3 hours at 63 °C in 20 ml of ExpressHybTMHybridization Solution (Clontech, Palo Alto, CA).
• Filters were hybridized in 15 ml hybridizing solution containing 1.6 x 10 6 cpm/ml of probe at 63°C overnight. Filters were washed at 55°C in 2X SSC, 0.1 % SDS and 1 mM EDTA and exposed to X- ray film at -80°C for 4 1/2 days. Thirteen positives were picked from the plates as plugs and placed in 1 ml LB +amp in 1.7 ml tubes. Tubes were placed at 4°C overnight. These 13 positives were subjected to two further rounds of purification. The tertiary plates were outgrown at 37°C after filter lifts were taken and single colonies were picked and sent to sequencing. Three of these were determined to contain sequence of the mouse ortholog of zcytoR 17.
• CTLL-2 is a mouse cytotoxic T lymphocyte cell line (ATCC No. TE3-214). This PCR reaction was ran as follows: 1 cycle at 95°C for 1 minute, 30 cycles at 95°C for 15 seconds, 68°C for 3 minutes, then 68°C for 10 minutes; 4°C soak. The PCR reaction used approximately 0.5 ng. of cDNA, 20 pmoles of each oligonucleotide, and 1 ⁇ l.
• PCR reaction was ran as follows: 30 cycles at 94°C for 45 seconds, 65°C for 45 seconds, 72°C for 1 minute, then 72°C for 7 minutes; 10°C soak. Most of the PCR reaction was loaded on a 1.0% agarose gel and the predominant band at approximately 360 bp was excised, the DNA fragment was eluted, and DNA sequencing was performed.
• mice zcytor 17 polynucleotide The sequence of the mouse zcytor 17 polynucleotide is shown in SEQ ED NO:56 and the corresponding amino acid sequence shown in SEQ ED NO:57.
• a truncated soluble form of the mouse zcytorl7 polynucleotide is shown in SEQ ED NO:92 and the corresponding amino acid sequence shown in SEQ ED NO:93.
• the panel was set up in a 96-well format that included a mouse genomic DNA (Clontech, Palo Alto, CA) positive control sample. Each well contained approximately 0.2-100 pg/ ⁇ l of cDNA.
• the PCR was set up using oligos ZC38,065 (SEQ HD NO:77) and ZC38,068 (SEQ ED NO:78), TaKaRa Ex TaqTM (TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye (Research Genetics, Inc., Huntsville, AL).
• the probe is cleaved by 5' nuclease activity of Taq polymerase which releases the reporter dye from the probe resulting in an increase in fluorescent emission.
• the primers and probes used for real-time quantitative RT-PCR analyses of Zcytorl7 expression were designed using the primer design software Primer ExpressTM (PE Applied Biosystems, Foster City, CA). Primers for human Zcytorl7 were designed spanning an intron-exon junction to eliminate amplification of genomic DNA.
• RNA samples (below) were screened for rRNA using a primer and probe set ordered from PE Applied Biosystems (cat# 4304483).
• the kit contains the rRNA forward primer
• the rRNA probe was labeled at the 5'end with a reporter fluorescent dye VIC (PE Applied Biosystems) and at the 3' end with the quencher fluorescent dye TAMRA (PE Applied Biosystems).
• the rRNA results also serve as an endogenous control and allow for the normalization of the Zcytorl7 mRNA expression results seen in the test samples.
• the CD19+ samples were isolated from human tonsil and activated with 0.5ug/ml ionomycin and lOng/ml PMA. Cells were then collected at 0, 4 hours and 24 hours and RNA isolated. Human CD14+ monocytes were activated with either 0. lug/ml LPS or l.Oug/ml LPS for 20 hours. Resting and activated cells were then collected and RNA isolated. In addition, RNA was isolated from resting and activated (lO.Oug/ml LPS) human monocyte cell lines HL-60, THP-1 (ATCC No. Tffi-202) and U937. THP-1 RNA was used as a control because it was shown to express Zcytor 17 by Northern Blot (Example 3).
• Relative levels of Zcytorl7 mRNA were determined by analyzing total RNA samples using the one-step RT-PCR method (PE Applied Biosystems). Total RNA from Zcytor 17 expressing THP-1 cells was isolated by standard methods and used to generate a standard curve used for quantitation. The curve consisted of 10-fold serial dilutions ranging from 0.25-0.00025ng/ ⁇ l for the rRNA screen and 250-0.25 ng/ ⁇ l for the Zcytorl7 screen with each standard curve point analyzed in triplicate. The total RNA samples from the human cells were also analyzed in triplicate for human Zcytorl7 transcript levels and for levels of rRNA as an endogenous control.
• Relative Zcytor 17 RNA levels were determined by using the Standard Curve Method as described by the manufacturer, PE Biosystems (User Bulletin #2: ABI Prism 7700 Sequence Detection System, Relative Quantitation of Gene Expression, December 11, 1997). The rRNA measurements were used to normalize the Zcytor 17 levels. Three experiments were done testing the aforementioned. Data shown in Tables 8 and 9 below are expressed as a ratio of Zcytorl7 mRNA to rRNA.

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